cDNA cloning and characterization of chalcone isomerase-fold proteins from snapdragon (Antirrhinum majus L.) flowers

Abstract

cDNAs coding for the chalcone isomerase-fold proteins (CIFP) of the snapdragon (Antirrhinum majus L.) were cloned and characterized. One of these CIFPs was a Cluster-1 member of the CIFP family, which was a catalytically active chalcone isomerase and thus termed AmCHI1. The recombinant AmCHI1 could act on 2′,4,4′,6′-tetrahydroxychalcone (THC) and 2′,3,4,4′,6′-pentahydroxychalcone (PHC) to produce naringenin and eriodictyol with k cat /K m values of 0.25 s −1 µM −1 and 0.071 s, respectively, at pH 7.5 and 4°C. The enzyme could not act on 4′-O-glucosides of THC and PHC. In the yellow snapdragon petals (cv. Yellow Butterfly), the temporal expression patterns of AmCHI1 were consistent with the observed temporal accumulation patterns of flavones. Thus, regulation of the AmCHI1 transcription and substrate specificity of the expressed AmCHI1 should serve as the key mechanisms that allows for partitioning of the flavonoid biosynthetic pathways into the aurones and the non-aurone flavonoids in snapdragon petal cells. The other CIFP cDNA, AmCIFP4, was a Cluster-4 member of the family and was similar in its primary structure to enhancers of the flavonoid production of torenia (Torenia×hybrida) and petunia (Petunia×hybrida). AmCIFP4 was more abundantly expressed than AmCHI1 irrespective of flower color.

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

“…Thus, a split‐ubiquitin (SU) system (Iyer et al ., ) was used to screen for interactions between these P450 proteins and other soluble flavonoid enzymes in snapdragon [i.e. CHS (AmCHS1 or Nivea) (Hatayama et al ., ); CHI (AmCHI1) (Fujino et al ., ); F3H (AmF3H or Incolorata); DFR (AmDFR or Pallida); and C4′GT (AmC4′GT) (Ono et al ., ); Figures and S1]. Because the coding sequence of the snapdragon anthocyanidin synthase gene ( ANS ) was not available in databases, ANS was not included in these interaction assays.…”

“…Binary interactions among torenia flavonoid enzymes [i.e. CHS, two isozymes of type‐1 CHI (CHI1_1 and CHI1_2; Fujino et al ., ), FNSII (Akashi et al ., ; Ueyama et al ., ), F3H, DFR, ANS, F3′H (Ueyama et al ., ) and F3′5′H] were comprehensively analyzed using the SU and Y2H systems (Figures and S6). The analyses of yeast growth and β‐galactosidase activity in the SU system (Figure ) revealed FNSII interacted with CHI1_1, CHI1_2, F3H and DFR (Figure a).…”

“…The complete open reading frames encoding the following flavonoid enzymes (DDBJ/ENA/GenBank accession numbers in parentheses) were cloned into the pGEM‐T Easy vector (Promega, Madison, WI, USA), and the nucleotide sequence of the inserted DNA was confirmed by sequencing: AmCHS1 (X03710) (Hatayama et al ., ), AmCHI1 (AB861648) (Fujino et al ., ), AmFNSII (AB028151) (Akashi et al ., ), AmF3H (LC194907), AmDFR (P14721) (Beld et al ., ), AmC4′GT (AB198665) (Ono et al ., ), AmAS1 (AB044884) (Nakayama et al ., ), AmF3 ′ H (DQ272592) (Schwinn et al ., ), ThCHS1 (AB106522) (Fukusaki et al ., ), ThCHI1_1 (LC194908), ThCHI1_2 (LC194909), ThFNSII (AB028152) (Akashi et al ., ), ThF3H (AB211958), ThDFR (AB012924) (Suzuki et al ., ), ThANS (identical to Torenia fournieri ANS; AB044091) (Nakamura et al ., ), THF3 ′ H (AB057672) (Ueyama et al ., ) and ThF3′5′H (AB012925) (Suzuki et al ., ). The AmF3H and AmDFR cDNAs were kindly provided by Dr Yuji Kishima, Hokkaido University.…”

“…To obtain anti‐AmCHS1 and anti‐AmCHI1 antibodies, electrophoretically homogeneous preparations of the non‐denatured forms of recombinant AmCHS1 (Hatayama et al ., ) and AmCHI1 (Fujino et al ., ) were used to immunize female Japanese white rabbits by an intradermal injection at multiple sites according to standard protocols (Vaitukaitis, ). To obtain anti‐AmFNSII and anti‐DFR antibodies, the peptides corresponding to amino acid residues 469–487 of AmFNS and residues 334–352 of AmDFR, which were predicted to serve as epitopes, were used to immunize female Japanese white rabbits.…”

Structure, expression profile and phylogenetic inference of chalcone isomerase-like genes from the narrow-leafed lupin (Lupinus angustifolius L.) genome

“…As the authors suggested, it could be related to the role of flavonoids in nodule development, as signal molecules during interactions with rhizobia. Recently, it was concluded that CHILs are enhancers of flavonoid production and flower pigmentation, and their function is conserved among diverse land plant species (Fujino et al, 2014 ; Morita et al, 2014 ).…”